DESCRIPTION (applicant?s abstract): Serine proteases are a class of enzymes that play critical roles in many human diseases, including cancer, cystic fibrosis, rheumatoid arthritis, emphysema, thromboembolic disease, hepatitis C, and cytomegalovirus. The design of highly specific serine protease inhibitors is a promising therapeutic approach for disease treatment; the potential of structure-based drug design has been amply demonstrated by the clinical success of HIV protease inhibitors in combating AIDS. In addition to serving as pharmaceutical targets, serine proteases also serve as a useful model for elucidating the strategies that enzymes use for catalysis. Despite all that is known about serine protease structure and mechanism, no solved structures are available for these enzymes complexed with their substrates. This missing piece of information stems from a technical difficulty; crystal structures present a molecular picture averaged over time, while enzymes complexed with their targets usually react too quickly to be captured by standard crystallographic methods. Developments in technology, including powerful synchrotron radiation sources and cryocrystallographic techniques, now have enabled structure determination for several other enzymes complexed with their natural substrates. The first aim of this proposal is to obtain a crystal structure for a representative serine protease, subtilisin, complexed with a specific substrate. The second aim is to use the methodology developed for the structure determination of the first subtilisin-substrate complex to obtain structures for a series of mutant subtilisin complexes. Structure-reactivity correlations will be drawn to clarify the importance of substrate orientation in catalysis. The final aim is to compare in detail the structure of a subtilisin-substrate complex with that of a subtilisin-protein inhibitor complex, to determine the structural features that make one complex reactive and the other unreactive. Because of the conservation of active site geometry and chemical mechanism in serine proteases, conclusions drawn about the orientation requirements for the hydrolysis reaction will be generalizable to all serine proteases. Taken together, the information provided by the successful completion of this proposal will provide an improved template for the future design of therapeutic inhibitors of enzymes in the serine protease family.